The specific aims of the proposed research are: 1) to finalize the structure of the functionalized non-heme iron center (H cluster) that is uniquely involved in both substrate (H2) activation and electron transfer; 2) to analyze the function of the H cluster and two ferredoxin- type clusters by specific probes utilizing biophysical techniques; 3) to attempt to isolate the gene product of the putative hydrogenase gene, hyd lambda from Desulfovibrio vulgaris which is suspected to be a new-type of (Fe) hydrogenase in Desulfovibrio and may resolve the problem of the number and types of (Fe) hydrogenases; 4) to establish a DNA transfer system with D. vulgaris using a wide host range plasmid and generate (Fe) hydrogenase negative mutants in order to obtain biosynthesis of active (Fe) hydrogenase and 5) to investigate the role of the small subunit and its putative signal peptide in the periplasmic localization of the (Fe) hydrogenase with the goal of determining how the large subunit that lacks a signal peptide is translocated across the cytoplasmic membrane.